This invention relates to compounds useful for the labeling of biological materials, such as DNA and proteins, and non-biological materials to make the materials fluorescent and easily detectable. In one embodiment, the compounds can be used to label and then sequence DNA after irradiation by light from a laser.
In one class of techniques for sequencing DNA, identical strands of DNA are marked with a fluorescent dye. The strands are marked by attaching specially synthesized fluorescent oligonucleotide primers or probes to the strands of DNA, or by attaching the fluorescent dye directly to the strands. The strands are separated into four aliquots. The strands in a given aliquot are either individually cleaved at or synthesized to any base belonging to only one of the four base types, which are adenine, guanine, cytosine, and thymine (hereinafter A, G, C and T). The adenine-, guanine-, cytosine-, and thymine-terminated strands are then electrophoresed for separation and the separated strands are irradiated by a laser and the emission from the fluorescent dye detected. The rate of electrophoresis indicates the DNA sequence.
Cyanine dyes are known to absorb far-red (600-700 nm) and near-infrared (700-1200 nm) light and techniques for the synthesis of derivatives of the cyanine dyes are known. It has been difficult, however, to obtain chromophores with absorption and emission bands that reduce the effect of background noise during gel electrophoresis when irradiating with a diode laser scanner. Some dyes, not described herein, exhibit absorption in wavelengths greater than 1200 nm (infrared) and also would provide discrimination against background noise.
Suitable types of cyanine dyes include heptamethine cyanine dyes. Cyanine dyes traditionally have been synthesized by a condensation reaction between a heterocyclic base containing an activated methyl group and an unsaturated bisaldehyde or its equivalent, usually a Schiff base in the presence of a catalyst. Sodium acetate has been used most frequently as a catalyst. In addition to ethanol, solvents such as acetic acid and/or acetic anhydride also have been commonly used, as in the synthesis of heptamethine pyrylium dyes.
This procedure suffers from several disadvantages, such as, for example: (1) the purification of the product is very difficult because of the side products due to aniline; (2) the use of a catalyst interferes with the purity of the product and warrants repeated purification; (3) the reaction is generally fast and cannot be employed for the synthesis of nonsymmetric dyes in one pot; and (4) the scaling up of the reaction products to larger gram quantities leads to several additional problems resulting in poor quality and yield.
The present invention describes new cyanine dyes, advantageous methods of making them, and the labeling of various materials with these dyes.
Accordingly, it is an object of the present invention to provide novel dyes that fluoresce in the far red, near infrared, or infrared region in selected wavelengths when attached to biological and nonbiological materials and that have sufficient quantum yield to make detection feasible.
It is a further object of the invention to provide a method of synthesizing new dyes having these characteristics.
It also is an object of the invention to provide a novel probe or primer containing a dye that fluoresces in the far red, near infrared, or infrared region.
It is a further object of the invention to provide a novel fluorescent marker, method of synthesizing the marker and method of attaching the marker to DNA and other biological and nonbiological material.
It is a further object of the invention to provide a novel technique for DNA sequencing.
In accordance with the present invention, novel cyanine dyes are provided which can be used to label biological molecules, such as DNA, proteins and antibodies, and non-biological molecules. The dyes have preferred spectra and high absorption and fluorescence properties. Each dye has at least one reactive group which enables it to be coupled easily to the molecule of interest.
The dyes of the invention have an absorption band and an emission band within a region encompassing the far red, near infrared, or infrared region when attached to a probe, primer, oligonucleotide or other molecule. The dyes are selected to provide high quantum yield in an optical band selected to reduce background noise. The preferred dyes for many applications calling for the labeling of biomolecules are cyanine dyes which have an NCS group, a carboxyl group or a hydroxy group. The NCS group reacts with the amino group of the biomolecule to form a thiourea linkage. A carboxyl group on the dye can be converted to an NHS ester that reacts with the amino group of the biomolecule to form a stable amide linkage, and a hydroxyl group on the dye can react with a biomolecule to form stable carbamate linkages through NHS carbonate ester activation.
The preferred dyes are heptamethine cyanines which efficiently absorb light having wavelengths in the region of 630 to 900 nm. These wavelengths are suitable for reducing background fluorescence in DNA sequencing and correspond to the radiation wavelengths of diode lasers made of such materials as GaAlAs, GaAs, InGaAlP, GaInP, AlGaAs, AlGaInP, GaAlP, InGaAsP, GaInP/AlInP, InGaP/InGaAlP, or GaInP/AlGaInP. The GaAlAs diode, for example, emits light at wavelengths in the region of 780-800 nm and is used for scanning the gel electrophoresis sandwich used for DNA sequencing.
The sequencing of far red, near infrared, and infrared fluorescent dye-labeled DNA and the detection of the DNA after irradiation by far red, near infrared, or infrared light from a laser diode can be readily accomplished using the novel compounds of this invention. The strands of DNA are continuously electrophoresed and identified for any of several purposes, such as, for example: (1) DNA sequencing and (2) analysis of strands varying in length as prepared by such techniques as restriction enzyme cutting or polymerase chain reaction (PCR).
To aid in identification, the strands are marked with fluorescent labels that emit light in the far red, near infrared, or infrared region. The strands are irradiated with light in the far red, near infrared, or infrared region and the light emitted from the fluorescent labels is detected and used to obtain information about the DNA strands.
The marking is accomplished by direct labeling of fluorescent markers to the strands or by fluorescently labeled probes or primers hybridized to the separated strands. The labeled strands are detected by scanning with a far red, near infrared, or infrared laser diode light source.